Basic study of corn protein, zein, as a biomaterial in tissue engineering, surface morphology and biocompatibility Dong, J., Q. Sun, et al. (2004), Biomaterials 25(19): 4691-7.Abstract: The zein films, were prepared for culturing human liver cells (HL-7702) and mice fibroblast cells (NIH3T3), while the Corning microplate and polylactic acid (PLA) were chosen as controls. The surface morphology of zein films prepared by two different methods was studied by scanning electron microscope (SEM), which revealed that the zein films were composed of particles of diameter 100-500 and 500-2500 nm, respectively. The biocompatibility of zein films was assessed by attachment, extensibility and proliferation of cells on them. Our study indicated that over 60% of both HL-7702 cells and NIH3T3 cells could attach to the Corning microplate, zein films and PLA at 3h after seeding. The concentration and particle sizes for preparing zein films did not seem to affect the proliferation of the cells tested. There were no significant differences in the proliferation of both HL-7702 cells and NIH3T3 cells between the Corning microplate and two kinds of zein films, except that the zein film composed of smaller particles at the lowest concentration exhibited a very good ability for proliferation of both the cells, while PLA was a poor matrix in the latter period of the cell proliferation. This preliminary study demonstrates that zein is a promising biomaterial with good biocompatibility for the development of tissue engineering.

Behavior of various orbital implants under axial compression Jordan, D. R., N. Ahuja, et al. (2005), Ophthal Plast Reconstr Surg 21(3): 225-9.Abstract: PURPOSE: To determine and compare the amount of force required to disrupt the integrity of various orbital implants. METHODS: Compression tests were carried out by using a servo-electrical universal testing system on orbital implants including aluminum oxide (Bioceramic implant, FCI, Issy-Les-Moulineaux, France), coralline hydroxyapatite (HA) (Bio-Eye, Integrated Orbital Implants, Inc., San Diego, CA, U.S.A.), bovine HA (Molteno M-Sphere, IOP Inc., Costa Mesa, CA, U.S.A.), synthetic HA (FCI3, FCI, Issy-Les-Moulineaux, France), Chinese HA (H + Y Comprehensive technologies, Philadelphia, PA, U.S.A.), polylactic acid (Kinsey Nash Corporation, Duluth, MN, U.S.A.), porous polyethylene (Medpor, Porex Surgical Inc., College Park, GA, U.S.A.), and polymethylmethacrylate. RESULTS: Two basic groups of implants were identified: those that eventually reach a critical compression point and collapse (coralline HA, aluminum oxide, synthetic FCI3 HA, bovine HA, Chinese HA, and polymethylmethacrylate), and those that do not collapse but gradually compress with increasing load (porous polyethylene, polylactic acid). For similar-sized implants, the critical collapse point was earliest for the FCI3 HA implant, followed by the coralline HA, aluminum oxide, and polymethylmethacrylate implants. Smaller-sized collapsible implants showed earlier critical collapse points than larger-sized implants of similar material. CONCLUSIONS: A technique was established to assess the force required to disrupt the integrity of various orbital implants that is reliable, unbiased, and repeatable with any orbital implant. Orbital implants of different materials and sizes demonstrate different degrees of integrity. It is important to use similar-sized implants when comparing the integrity of different implant materials because size influences the force required to overcome the structural integrity of the implant.

Behaviour of human endothelial cells on surface modified NiTi alloy Plant, S. D., D. M. Grant, et al. (2005), Biomaterials 26(26): 5359-67.Abstract: Intravascular stents are being designed which utilise the shape memory properties of NiTi alloy. Despite the clinical advantages afforded by these stents their application has been limited by concerns about the large nickel ion content of the alloy. In this study, the surface chemistry of NiTi alloy was modified by mechanical polishing and oxidising heat treatments and subsequently characterised using X-ray photon spectroscopy (XPS). The effect of these surfaces on monolayer formation and barrier integrity of human umbilical vein endothelial cells (HUVEC) was then assessed by confocal imaging of the adherens junctional molecule VE-cadherin, perijunctional actin and permeability to 42kDa dextrans. Dichlorofluoroscein assays were used to measure oxidative stress in the cells. XPS analysis of NiTi revealed its surface to be dominated by TiO(2). However, where oxidation had occurred after mechanical polishing or post polishing heat treatments at 300 and 400 degrees C in air, a significant amount of metallic nickel or nickel oxide species (10.5 and 18.5 at%) remained on the surface. Exposure of HUVECs to these surfaces resulted in increased oxidative stress within the cells, loss of VE-cadherin and F-actin and significantly increased paracellular permeability. These pathological phenomena were not found in cells grown on NiTi which had undergone heat treatment at 600 degrees C. At this temperature thickening of the TiO(2) layer had occurred due to diffusion of titanium ions from the bulk of the alloy, displacing nickel ions to sub-surface areas. This resulted in a significant reduction in nickel ions detectable on the sample surface (4.8 at%). This study proposes that the integrity of human endothelial monolayers on NiTi is dependent upon the surface chemistry of the alloy and that this can be manipulated, using simple oxidising heat treatments.

beta(2)-Microglobulin-selective direct hemoperfusion column for the treatment of dialysis-related amyloidosis Kutsuki, H. (2005), Biochim Biophys Acta 1753(1): 141-5.Abstract: Lixelle is a direct hemoperfusion-type adsorption column that was developed to selectively eliminate beta2-microglobulin (beta2-m) from the circulating blood of patients with dialysis-related amyloidosis (DRA). The adsorbent in Lixelle comprises porous cellulose beads to which hydrophobic hexadecyl alkyl chain is covalently bound. One milliliter of wet Lixelle beads eliminates more than 1 mg of beta2-m in vitro. In hemodialysis patients who were treated with Lixelle, Lixelle improved joint pain, nocturnal awakening, pinch strength, motor terminal latency, and their activity of daily living. The adsorbent adsorbs beta2-m selectively but not specifically, as well as inflammatory cytokines such as interleukin-1beta and IL-6 which are considered to be involved in the development of DRA. Lixelle treatments reduce the circulating levels of beta2-m and inflammatory cytokines, thereby improving the symptoms of patients with DRA.

Better osteoblast adhesion on nanoparticulate selenium- A promising orthopedic implant material Perla, V. and T. J. Webster (2005), J Biomed Mater Res A 75(2): 356-64.Abstract: Apart from problems such as poor osseointegration, stress shielding, and wear debris-associated bone cell death, a major concern of metallic orthopedic implants is that they slowly corrode under in vivo environments. It is possible that continuous tissue exposure to metallic corrosion products limits orthopedic implant efficacy; this is especially true for patients receiving implants due to bone cancer. To date, there is no metallic orthopedic implant available in the market that specifically deals with the prevention and/or recurring cancer that may happen in these patients. The objective of this study was to deal with these problems in an integrated way by introducing a new biomaterial to the orthopedic community with anticancer chemistry: selenium (Se). In this study, six types of Se compacts were tested for bone cell (osteoblast) adhesion under in vitro conditions. Two types of cylindrical compacts were made with conventional Se metal particles in the micron (6.539 +/- 1.364-microm diameter) and submicron (0.963 +/- 0.139-microm diameter) range. These two types of compacts were chemically etched with different concentrations of NaOH to create two additional types of Se particles in each category: conventional size particles with nanosurface roughness and nanometer particles (0.204- to 0.264-microm diameter). Results showed for the first time, enhanced osteoblast adhesion on particulate surfaces of the compacts made from conventional Se compared with reference nonparticulate wrought titanium sheets. More importantly, this study provided the first evidence that osteoblast density was further increased on the surfaces of the Se compacts with nanometer particles. These initial findings indicate that there may be a promising future for nanoparticulate Se as an anticancer biocompatible orthopedic material.

Biaxial failure properties of planar living tissue equivalents Billiar, K. L., A. M. Throm, et al. (2005), J Biomed Mater Res A 73(2): 182-91.Abstract: Quantification of the mechanical properties of living tissue equivalents (LTEs) is essential for assessing their ultimate functionality as tissue substitutes, yet their delicate nature makes failure testing problematic. For this study, we evaluated the validity of using an inflation device for quantifying the biaxial tensile failure properties of extremely delicate fibroblast-populated collagen gels (CGs) and fibrin gels (FGs). Small samples were circularly clamped and then inflated until rupture. Each sample assumed an approximately spherical shape and burst at its center indicating effective clamping. After two weeks in culture, all LTEs tested were fragile, but the FGs were significantly stronger and more extensible than the CGs (ultimate tensile strength 6.0 kPa +/- 2.0 kPa vs. 2.8 kPa +/- 0.7 kPa; failure strain 3.5 +/- 0.9 vs. 0.26 +/- 0.05, n = 4). After an additional 11 days of culture, the strength of the FGs increased significantly (26.5 kPa +/- 12.7 kPa), and the extensibility decreased (1.9 +/- 0.8, n = 3). This study demonstrates that subtle differences in the properties of LTEs can be measured using inflation methods with minimal sample handling and without having to grow the tissues into anchors or cut the specimens.

Biaxial mechanical response of bioprosthetic heart valve biomaterials to high in-plane shear Sun, W., M. S. Sacks, et al. (2003), J Biomech Eng 125(3): 372-80.Abstract: Utilization of novel biologically-derived biomaterials in bioprosthetic heart valves (BHV) requires robust constitutive models to predict the mechanical behavior under generalized loading states. Thus, it is necessary to perform rigorous experimentation involving all functional deformations to obtain both the form and material constants of a strain-energy density function. In this study, we generated a comprehensive experimental biaxial mechanical dataset that included high in-plane shear stresses using glutaraldehyde treated bovine pericardium (GLBP) as the representative BHV biomaterial. Compared to our previous study (Sacks, JBME, v.121, pp. 551-555, 1999), GLBP demonstrated a substantially different response under high shear strains. This finding was underscored by the inability of the standard Fung model, applied successfully in our previous GLBP study, to fit the high-shear data. To develop an appropriate constitutive model, we utilized an interpolation technique for the pseudo-elastic response to guide modification of the final model form. An eight parameter modified Fung model utilizing additional quartic terms was developed, which fitted the complete dataset well. Model parameters were also constrained to satisfy physical plausibility of the strain energy function. The results of this study underscore the limited predictive ability of current soft tissue models, and the need to collect experimental data for soft tissue simulations over the complete functional range.

Biaxial testing of membrane biomaterials: testing equipment and procedures Nielsen, P. M., P. J. Hunter, et al. (1991), J Biomech Eng 113(3): 295-300.Abstract: A testing facility for measuring the biaxial mechanical properties of highly deformable membranes is described. Forces are applied, via strain-gauge force transducers, to four points on each side of an initially square 12 to 25 mm membrane sample to produce biaxial extensions of up to 80 percent of undeformed length. Strain is estimated from the displacement of markers bounding a 1 to 2 mm central square. The accuracy of stress and strain field measurements has been assessed by finite element analysis of a biaxially-loaded isotropic elastic membrane. Major advantages of the present system over those previously described in the literature are that 1) sample mounting procedures are simplified, 2) there is provision for independent adjustment of stress field uniformity and measurement of the applied point forces and 3) faster strain rates can be imposed on the relatively small samples tested.

Bilirubin removal from human plasma by Cibacron Blue F3GA using immobilized microporous affinity membranous capillary method Zhang, L. and G. Jin (2005), J Chromatogr B Analyt Technol Biomed Life Sci 821(1): 112-21.Abstract: A novel affinity sorbent system for direct bilirubin removal from human plasma was developed. These new adsorbents comprise Cibacron Blue F3GA as the specific ligand, and microporous membranous poly(tetrafluoroethylene) capillary (modified by coating with a hydrophilic layer of poly(vinyl alcohol) after activation) as the carrier matrix. The affinity adsorbents carrying 126.5 micromol Cibacron Blue F3GA/g polymer was then used to remove bilirubin in a flow-injection system. Non-specific adsorption on the poly(vinyl alcohol) coated capillary remains low, and higher affinity adsorption capacity, of up to 76.2 mg/g polymer was obtained after dye immobilization. The bilirubin adsorption capacity of the affinity capillary decreased with increase in the recirculation rate of plasma. The adsorption capacity increased with increase the temperature while decreased with increase the ionic strength. The maximum adsorption was only observed in neutral solution (pH 6-7). The adsorption isotherm fitted the Langmuir model well. These new adsorbents have higher velocity of mass transfer, better adsorption capacity, less fouling, longer service life and good reusability. The results of blood tests suggested the dye affinity capillary has good blood compatibility.

Binary immobilization of tyrosinase by using alginate gel beads and poly(acrylamide-co-acrylic acid) hydrogels Yahsi, A., F. Sahin, et al. (2005), Int J Biol Macromol 36(4): 253-8.Abstract: The use of the immobilized and the stable enzymes has immense potential in the enzymatic analysis of clinical, industrial and environmental samples. However, their widespread uses are limited due to the high cost of their production. In this study, binary immobilization of tyrosinase by using Ca-alginate and poly(acrylamide-co-acrylic acid) [P(AAm-co-AA)] was investigated. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the free and binary immobilized enzymes. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized tyrosinase were also examined. For the free and binary immobilized enzymes on Ca-alginate and P(AAm-co-AA), optimum pH was found to be 7 and 5, respectively. Optimum temperature of the free and immobilized enzymes was observed to be 30 and 35 degrees C, respectively. Reuse number, storage and thermal stability of the free tyrosinase were increased by a result of binary immobilization.

Binding and orientation of fibronectin on surfaces with collagen-related peptides. Student Research Award in the Masters Science Degree Candidate category, 27th annual meeting of the Society for Biomaterials, St. Paul, MN, April 24-29, 2001 Klueh, U., S. Goralnick, et al. (2001), J Biomed Mater Res 56(3): 307-23.Abstract: Although fibronectin (FN) has been used in a variety of in vitro studies to enhance cell and bacteria adhesion, relatively little is known about the molecular interactions of FN with surfaces, particularly the interactions that can control the binding, conformation, and functionality of FN on these surfaces. Even less is known about approaches needed to control binding, orientation, and functionality of FN bound on surfaces. To begin to fill this gap in our knowledge, we hypothesized that functional FN can be bound and specifically oriented on polystyrene surfaces with FN-specific collagen-related peptides (CRPs). We further hypothesized that monoclonal antibodies that react with specific epitopes on FN can be used to quantify both FN binding and orientation on these surfaces. On the basis of these hypotheses, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces with CRPs. To bind FN to surfaces, we used two different CRPs: CRP-I (TLQPVYEYMVGV) and CRP-II (TGLPVGVGYVVTVLT). The binding and orientation of the FN molecule to these immobilized CRPs was quantified with (125)I-FN and monoclonal antibodies. Monoclonal antibodies used for this study were reactive with specific regions of the FN molecule, that is, the amino (N) terminus (anti-N antibodies) and carboxyl (C) terminus (anti-C antibodies). The results of our studies demonstrated that although CRP-I and CRP-II could be bound directly to polystyrene, these directly immobilized CRPs failed to bind (125)I-FN. Thus, to facilitate FN binding to the CRPs, we used bovine serum albumin (BSA) as a spacer to physically elevate the CRPs away from the polystyrene surface. Thus, CRP-I and CRP-II were covalently linked to BSA via the N and C termini of each CRP (CRP-I-BSA and CRP-II-BSA). (125)I-CRP-BSAs were all found to bind to equivalent levels on polystyrene (1.60-2.60 microg/cm2). When CRP-BSAs were immobilized on polystyrene, they all successfully bound (125)I-FN in a range of 34-72 ng/cm2 (mean). Using monoclonal antibodies to FN to characterize the orientation of FN bound to the various CRP-BSAs, we demonstrated that (1) FN consistently bound to either CRP-I-BSA or CRP-II-BSA; (2) bound FN reacted significantly more with anti-C antibodies than with anti-N antibodies; and (3) the increased reactivity of bound FN to anti-C antibodies was consistent, whether FN was bound by CRP-I or CRP-II or the CRPs were bound to BSA by the C or N termini. These data demonstrated an enhanced binding of anti-C antibodies to immobilized CRP-BSA relative to anti-N antibodies. We interpreted the data to be the result of FN binding in an oriented fashion with N termini of FN bound tightly to the BSA-polystyrene surface. In this position, the C termini of FN are exposed and available for binding by the anti-C antibodies. Alternatively, in this orientation the N termini of the FN would not be available to bind the anti-N antibodies, thereby explaining the decreased reactivity of the CRP immobilized FN to the anti-N antibodies. These studies not only demonstrate the utility of peptides in binding and orienting large molecular weight proteins such as FN on surfaces but underscore the need for well-characterized reagents (e.g., monomeric/functional FN and antibodies) to specifically bind, orient, and characterize large molecular weight proteins immobilized on various surfaces.

Binding of a model regulator of complement activation (RCA) to a biomaterial surface: surface-bound factor H inhibits complement activation Andersson, J., R. Larsson, et al. (2001), Biomaterials 22(17): 2435-43.Abstract: The complement system is an important inflammatory mediator during procedures such as cardiopulmonary bypass and hemodialysis when blood is exposed to large areas of biomaterial surface. This contact between blood and the biomaterials of implants and extracorporeal circuits leads to an inflammatory response mediated by the complement system. The aim of this study was to assess the ability of a complement regulator (factor H) immobilised on a biomaterial surface to inhibit complement cascade mediated inflammatory responses. The cross-linker N-succinimidyl 3-(2-pyridyldithio) propionate was used to immobilise factor H on a model biomaterial surface without affecting the biological activity of the inhibitor. Binding of factor H was then characterised using quartz crystal microbalance-dissipation (QCM-D) and enzyme immunoassays for products of complement activation: bound C3 fragments and soluble C3a, sC5b-9, and C1s-C1INA. Immobilised factor H reduced the amount C3 fragments deposited on the biomaterial surface after incubation with serum, plasma. or whole blood. In addition, lower levels of soluble C3a and sC5b-9 were generated after incubation with whole blood. In summary, we have demonstrated that complement activation on a highly activating model surface can be inhibited by immobilised factor H and have defined prerequisites for the preparation of future biomaterial surfaces with immobilised regulators of complement activation.

Binding of C3 fragments on top of adsorbed plasma proteins during complement activation on a model biomaterial surface Andersson, J., K. N. Ekdahl, et al. (2005), Biomaterials 26(13): 1477-85.Abstract: In the present study we investigate whether complement activation in blood in contact with a model biomaterial surface (polystyrene) occurs directly on the material surface or on top of an adsorbed plasma protein layer. Quartz crystal microbalance-dissipation analysis (QCM-D) complemented with enzyme immunoassays and Western blotting were used. QCM-D showed that the surface was immediately covered with a plasma protein film of approximately 8 nm. Complement activation that started concomitantly with the adsorption of the protein film was triggered by a self-limiting classical pathway activation. After adsorption of the protein film, alternative pathway activation provided the bulk of the C3b deposition that added 25% more mass to the surface. The build up of alternative pathway convertase complexes using purified C3 and factors B and D on different protein films as monitored by QCM-D showed that only adsorbed albumin, IgG, but not fibrinogen, allowed C3b binding, convertase assembly and amplification. Western blotting of eluted proteins from the material surface demonstrated that the C3 fragments were covalently bound to other proteins. This is consistent with a model in which the activation is triggered by initiating convertases formed by means of the initially adsorbed proteins and the main C3b binding is mediated by the alternative pathway on top of the adsorbed protein film.

Binding of mannose-functionalized dendrimers with pea (Pisum sativum) lectin Schlick, K. H., R. A. Udelhoven, et al. (2005), Mol Pharm 2(4): 295-301.Abstract: Lectins are invaluable tools for chemical biology because they recognize carbohydrate arrays. Multivalent carbohydrate binding by lectins is important for processes such as bacterial and viral adhesion and cancer metastasis. A better understanding of mammalian lectin binding to carbohydrate arrays is critical for controlling these and other cellular recognition processes. Plant lectins are excellent model systems for the study of multivalent protein-carbohydrate interactions because of their robustness and ready availability. Here, we describe binding studies of mannose-functionalized poly(amidoamine) (PAMAM) dendrimers to a mitogenic lectin from Pisum sativum (pea lectin). Hemagglutination and precipitation assays were performed, and results were compared to those obtained from concanavalin A (Con A), a lectin that has been studied in more detail. Isothermal titration calorimetry (ITC) experiments are also described.

Bioabsorbable fixation devices in trauma and bone surgery: current clinical standing Waris, E., Y. T. Konttinen, et al. (2004), Expert Rev Med Devices 1(2): 229-40.Abstract: Bioabsorbable fixation devices are increasingly used in trauma, orthopedic and craniomaxillofacial surgery. The devices are essentially made of polylactic acid and/or polyglycolic acid polymers. Ultra-high-strength implants are manufactured from such polymers using self-reinforcing techniques. Implants are available for stabilization of fractures, osteotomies, bone grafts and fusions, as well as for reattachment of ligaments, tendons, meniscal tears and other soft tissue structures. As these implants are completely absorbed, the need for a removal operation is overcome and long-term interference with tendons, nerves and the growing skeleton is avoided. The risk of implant-associated stress shielding, peri-implant osteoporosis and infections is reduced. Implants do not interfere with clinical imaging. Current clinical use of bioabsorbable devices is reviewed.

Bioactivation of an anorganic bone matrix by P-15 peptide for the promotion of early bone formation Thorwarth, M., S. Schultze-Mosgau, et al. (2005), Biomaterials 26(28): 5648-57.Abstract: This animal experiment compared the regenerative processes within defined bony defects of the porcine skull after delivery of routinely utilized bone graft materials: anorganic bone matrix (ABM) and an identical ABM carrying the cell binding peptide P-15. Particulated autogenous bone was used as a control group. The chosen porcine model guaranteed the transferability of the obtained results to clinical practice. A total observation period of 6 months was defined. The bone samples were examined microradiographically and histologically at 8 specific times. Sufficient osseointegration and osseoconduction could be demonstrated for both anorganic bone minerals. However, in the selected model significantly higher mineralization rates (p = 0.0286) were found in the microradiographic image at 12 weeks after application of the bioactive form. The histological examination confirmed this accelerating effect on bone formation starting at day 3. At the end of the study after 6 months, the mineralization values had equalized in both study groups. For the first time, the material was demonstrated to be suitable as a bone substitute material for the treatment of larger bony defects in a large animal model. The P-15 sequence accelerated the process of bone formation on the surface of the anorganic bone matrix as early as 3 days but was not traced over the whole term of the study.

Bioactivation of inert alumina ceramics by hydroxylation Fischer, H., C. Niedhart, et al. (2005), Biomaterials 26(31): 6151-7.Abstract: Alumina ceramics (Al(2)O(3)) are frequently used for medical implants and prostheses because of the excellent biocompatibility, and the high mechanical reliability of the material. Inauspiciously alumina is not suitable for implant components with bone contact, because the material is bioinert and thereby no bony ongrowth, and subsequently loosening of the implant occurs. Here, we present a new method to bioactivate the surface of the material. Specimens made of high purity alumina were treated in sodium hydroxide. Cell culture tests with osteoblast-like cells as well as spectroscopical and mechanical tests were performed. Aluminium hydroxide groups were detected on the surface of the treated specimens. Enhanced cell adhesion, proliferation and secretion of osteocalcin were determined after hydroxylation. The bioactivating treatment had no deteriorating effect on the short- and long-term strength behaviour. Our results indicate that the described surface technique could be used to develop a new class of osseointegrative high-strength ceramic implants.